The goals of this research are to reconstitute the initiation of transcription by yeast RNA polymerase II from purified components, to determine the mechanism of initiation, and to elucidate the mechanisms of positive and negative control. These goals will be accomplished by the fractionation of yeast extracts we have developed that support accurately initiated and appropriately regulated transcription. We have so far resolved five fractions required for accurate initiation of transcription. A major objective is now to purify the five or more essential components of the reaction to homogeneity in amounts sufficient for mechanistic studies. Straightforward fractionation will be augmented by additional approaches, such as the use of antibodies raised by others against yeast RNA polymerase 11 and against mammalian initiation and elongation factors to assay specifically for these components. A mammalian activator, herpes VP16, and a yeast activator, a T-rich binding factor, have given large enhancements of transcription in our in vitro system. We will characterize these effects with regard to range, cooperativity, and so forth, and we will identify the component(s) of the transcription system with which the activators interact. We will also pursue a GAL4-GAL80 complex we have isolated for use as an inducible activator in the transcription system. We previously identified two yeast proteins, referred to as GRFI and ABFI, which bind specifically to and mediate the effects of the mating type locus transcriptional silencer. GRFI on its own serves to activate transcription of many yeast genes in vivo and also recognizes the repeated sequence at yeast telomeres, while ABFI recognizes a sequence found at most ARS elements. GRFI gives a modest enhancement of transcription in vitro. We will pursue this effect, to determine what is limiting, and will also try to reconstitute silencer action in vitro. We hope to find the connections between enhancer, silencer, ARS, and telomere function implicit in the involvement of common proteins.